The mammalian immune system has evolved a variety of mechanisms to protect the host from cancerous cells. An important component of this response is mediated by cells referred to as T cells. Cytotoxic T lymphocytes (CTL) are specialized T cells that primarily function by recognizing and killing cancerous cells or infected cells, but they can also function by secreting soluble molecules referred to as cytokines that can mediate a variety of effects on the immune system. T helper cells primarily function by recognizing antigen on specialized antigen presenting cells, and in turn secreting cytokines that activate B cells, T cells, and macrophages. A variety of evidence suggests that immunotherapy designed to stimulate a tumor-specific CTL response would be effective in controlling cancer. For example, it has been shown that human CTL recognize sarcomas (Slovin et al., 1986), renal cell carcinomas (Schendel et al., 1993), colorectal carcinomas (Jacob et al., 1997), ovarian carcinomas (Peoples et al., 1993), pancreatic carcinomas (Peiper et al., 1997), squamous tumors of the head and neck (Yasumura et al., 1993), and squamous carcinomas of the lung (Slingluff et al., 1994; Yoshino et al., 1994). The largest number of reports of human tumor-reactive CTLs, however, has concerned melanomas (Boon et al., 1994). The ability of tumor-specific CTL to mediate tumor regression, in both human (Parmiani et al., 2002; Weber, 2002) and animal models, suggests that methods directed at increasing CTL activity would likely have a beneficial effect with respect to tumor treatment.
Melanoma, or skin cancer, is a disease that is diagnosed in approximately 54,200 persons per year. Conventional therapy for the disease includes surgery, radiation therapy, and chemotherapy. In spite of these approaches to treatment, approximately 7,600 individuals die in the United States every year due to melanoma. Overall, the 5-year survival rate for the disease is 88%. The survival rate drops, however, in more advanced stages of the disease with only about 50% of Stage III patients and 20-30% of Stage IV patients surviving past five years. In patients where the melanoma has metastasized to distant sites, the 5-year survival dips to only 12%. Clearly, there is a population of melanoma patients that is in need of better treatment options. More recently, in an attempt to decrease the number of deaths attributed to melanoma, immunotherapy has been added to the arsenal of treatments used against the disease.
Dramatic regressions of melanoma have been induced with several types of immune therapies, including high-dose interleukin-2 and anti-CTLA4 antibody, which is FDA approved therapies for advanced melanoma, and adoptive T cell therapy, with reported objective response rates of 17%, 13%, and 51%, respectively, and with complete response (CR) rates in the range of 4-7%. Results with these therapies provide proof-of-principle for the therapeutic potential of immune therapy in melanoma. Unfortunately, the toxicities for all three therapies limit participant eligibility; so less toxic immune therapies with vaccines are being explored as alternative treatment options. This is especially true in the adjuvant setting where the only FDA-approved adjuvant therapy for patients with resected high-risk melanoma is high-dose, systemic interferon alpha. However, the most recent pooled analysis of interferon alpha therapy highlights the questionable survival advantage even of that therapy, for patients in the adjuvant setting. Thus, there is a critical need for additional new therapies for melanoma, both for adjuvant therapy of high-risk resected melanoma and for therapy of patients who are not candidates for, or fail, other therapies in the setting of advanced disease.
In order for CTL to kill or secrete cytokines in response to a cancer cell, the CTL must first recognize the cancer cell (Townsend & Bodmer, 1989). This process involves the interaction of the T cell receptor, located on the surface of the CTL, with what is generically referred to as an MHC-peptide complex which is located on the surface of the cancerous cell. MHC (major histocompatibility-complex)-encoded molecules have been subdivided into two types, and are referred to as class I and class II MHC-encoded molecules. In the human immune system, MHC molecules are referred to as human leukocyte antigens (HLA). Within the MHC complex, located on chromosome six, are three different loci that encode for class I MHC molecules. MHC molecules encoded at these loci are referred to as HLA-A, HLA-B, and HLA-C. The genes that can be encoded at each of these loci are extremely polymorphic, and thus, different individuals within the population express different class I MHC molecules on the surface of their cells. HLA-A1, HLA-A2, HLA-A3, HLA-B7, HLA-B14, HLA-B27, and HLA-B44 are examples of different class I MHC molecules that can be expressed from these loci.
The peptides which associate with the MHC molecules can either be derived from proteins made within the cell, in which case they typically associate with class I MHC molecules (Rock & Goldberg, 1999); or they can be derived from proteins which are acquired from outside of the cell, in which case they typically associate with class II MHC molecules (Watts, 1997). The peptides that evoke a cancer-specific CTL response most typically associate with class I MHC molecules. The peptides themselves are typically nine amino acids in length, but can vary from a minimum length of eight amino acids to a maximum of fourteen amino acids in length. Tumor antigens may also bind to class II MHC molecules on antigen presenting cells and provoke a T helper cell response. The peptides that bind to class II MHC molecules are generally twelve to nineteen amino acids in length, but can be as short as ten amino acids and as long as thirty amino acids.
The process by which intact proteins are degraded into peptides is referred to as antigen processing. Two major pathways of antigen processing occur within cells (Rock & Goldberg, 1999). One pathway, which is largely restricted to professional antigen presenting cells such as dendritic cells, macrophages, and B cells, degrades proteins that are typically phagocytosed or endocytosed into the cell. Peptides derived from this pathway can be presented on either class I or to class II MHC molecules. A second pathway of antigen processing is present in essentially all cells of the body. This second pathway primarily degrades proteins that are made within the cells, and the peptides derived from this pathway primarily bind to class I MHC molecules. Antigen processing by this latter pathway involves polypeptide synthesis and proteolysis in the cytoplasm, followed by transport of peptides to the plasma membrane for presentation. These peptides, initially being transported into the endoplasmic reticulum of the cell, become associated with newly synthesized class I MHC molecules and the resulting complexes are then transported to the cell surface. Peptides derived from membrane and secreted proteins have also been identified. In some cases these peptides correspond to the signal sequence of the proteins which is cleaved from the protein by the signal peptidase. In other cases, it is thought that some fraction of the membrane and secreted proteins are transported from the endoplasmic reticulum into the cytoplasm where processing subsequently occurs. Once bound to the class I MHC molecule, the peptides are recognized by antigen-specific receptors on CTL. Several methods have been developed to identify the peptides recognized by CTL, each method of which relies on the ability of a CTL to recognize and kill only those cells expressing the appropriate class I MHC molecule with the peptide bound to it. Mere expression of the class I MHC molecule is insufficient to trigger the CTL to kill the target cell if the antigenic peptide is not bound to the class I MHC molecule. Such peptides can be derived from a non-self source, such as a pathogen (for example, following the infection of a cell by a bacterium or a virus) or from a self-derived protein within a cell, such as a cancerous cell. The tumor antigens from which the peptides are derived can broadly be categorized as differentiation antigens, cancer/testis antigens, mutated gene products, widely expressed proteins, viral antigens and most recently, phosphopeptides derived from dysregulated signal transduction pathways. (Zarling et al., 2006).
Adoptive T cell therapy of melanoma is described in two recent publications: Dudley et al., 2008 and Rosenberg & Dudley, 2009. For adoptive T cell therapy, late stage metastatic melanoma patients are treated as if they were undergoing an organ transplant operation. Tumor is resected and cytotoxic T cells that have infiltrated the tumor are harvested and exposed to a particular class I peptide antigen (MART-1). Those that recognize this antigen are then allowed to expand until the total number of MART-1 specific cells reach 100 billion. The patient receives whole body irradiation and chemotherapy to wipe out 98% of his/her immune system. The MART specific T cells are then given back to the patient and circulate throughout the body looking for tumor. In the most recent clinical trial, tumors in 72% of the patients showed objective responses with this therapy at all sites of metastasis including lymph nodes, bone, lung, liver, and brain. Twenty-eight percent of the patients had complete remission of the disease.
Immunization with melanoma-derived, class I or class II MHC-encoded molecule associated peptides, or with a precursor polypeptide or protein that contains the peptide, or with a gene that encodes a polypeptide or protein containing the peptide, are forms of immunotherapy that can be employed in the treatment of melanoma. Identification of the immunogens is a necessary first step in the formulation of the appropriate immunotherapeutic agent or agents. Although a large number of tumor-associated peptide antigens recognized by tumor reactive CTL have been identified, there are few examples of antigens that are derived from proteins that are selectively expressed on a broad array of tumors, as well as associated with cellular proliferation and/or transformation.
Attractive candidates for this type of antigen are peptides derived from proteins that are differentially phosphorylated on serine (Ser), threonine (Thr), and tyrosine (Tyr). See Zarling et al., 2000. Due to the increased and dysregulated phosphorylation of cellular proteins in transformed cells as compared to normal cells, tumors are likely to present a unique subset of phosphorylated peptides on the cell surface that are available for recognition by cytotoxic T-lymphocytes (CTL). Presently, there is no way to predict which protein phosphorylation sites in a cell will be unique to tumors, survive the antigen processing pathway, and be presented to the immune system in the context of 8-14 residue phosphopeptides bound to class I MHC molecules.
Thirty-six phosphopeptides were disclosed as presented in association with HLA A*0201 on cancer cells. (see Table 1 of Zarling et al., 2006). Parent proteins for four of these peptides—beta-catenin, insulin receptor substrate-2 (IRS-2), tensin-3, and Jun-C/D—are associated with cytoplasmic signaling pathways and cellular transformation.
While both normal and cancer cells lines express the parent proteins, only the three cancer lines express phosphorylated class I peptide sequences within IRS-2 and beta-catenin, respectively. Mice expressing a transgenic recombinant human A*0201 MHC molecule were immunized with a synthetic class I phosphopeptides from IRS-2 and beta-catenin that were pulsed onto activated bone-marrow derived dendritic cells. Cytotoxic T cells were generated that recognized all three cancer cell lines but not the control JY cell line (i.e., an Epstein-Barr virus transformed B lymphoblastoid cell line).
β-catenin, a protein involved in cell adhesion and a downstream mediator of Wnt signaling, has been implicated in tumor development and progression (Takemaru et al., 2008). An HLA-A*0201-restricted phosphorylated peptide derived from β-catenin and (residues 30-39) that is presented by melanoma cell lines was described by (Zarling et al., 2006). Mutations in this region of β-catenin or in “destruction complex” proteins diminish phosphorylation and degradation of β-catenin and thereby stabilize the protein (Yost et al., 1996). Once stabilized, β-catenin translocates into the nucleus by an unknown mechanism where it associates with TCF/Lef proteins to activate transcription of genes such as cyclin D1 (Tetsu & McCormick, 1999), c-myc (He et al., 1998), and metalloproteases (Crawford et al., 1999; Takahashi et al., 2002), which promote tumorigenesis and metastasis.
While mutations in β-catenin or the destruction complex proteins are involved in the development of gastrointestinal cancers (Morin et al., 1997; Ogasawara et al., 2006), they are rarely found in human melanoma samples (Rimm et al., 1999; Omholt et al., 2001; Worm et al., 2004) and cell lines (Pollock & Hayward, 2002; Worm et al., 2004). Additionally, the expression of β-catenin in melanoma cells diminishes with disease progression (Sanders et al., 1999; Kageshita et al., 2001; Maelandsmo et al., 2003; Krengel et al., 2004; Hoek et al., 2006; Pecina-Slaus et al., 2007). Despite the reduced expression, nuclear β-catenin has been observed in melanoma samples and may be transcriptionally active in promoting invasive behavior of melanoma cells (Rimm et al., 1999; Bachmann et al., 2005; Chien et al., 2009; Arozarena et al., 2011).
Degradation of β-catenin is dependent on phosphorylation of the protein at S33, S37, and T41 by GSK-3β (Kimelman & Xu, 2006). Thus detection of this phosphorylated form of the protein in cells indicates that β-catenin has been marked for degradation. Phosphorylated β-catenin has been detected in metastatic melanomas and to a lesser extent, primary melanomas (Kielhorn et al., 2003) but the relative abundances of the different forms of phosphorylated β-catenin (S33/S37/T41, S37/T41, S33/S37, T41, S37, or S33 only) were not distinguished.
Until the present disclosure, no studies have examined MHC class-I-bound phosphopeptide displayed on primary human tumor samples, and there is only limited evidence of a human immune response against class-I restricted phosphopeptides.